Cytotoxic T lymphocytes (CTLs) play a central role in cellular immune responses by destroying infected or transformed target cells. Their potent anti-tumor activity has made them the centerpiece of several promising immunotherapeutic strategies to fight cancer. CTLs operate by forming a close, radially symmetric contact with their target cell known as an immunological synapse (IS). Then, they secrete cytolytic molecules into the synaptic space to induce target cell death. It is generally thought that the cytoskeletal framework of the IS potentiates this response. Cortical filamentous actin (F-actin) is enriched in the periphery of the IS and depleted from the center, forming a characteristic ring. Concomitantly, the centrosome, which serves as a focal point for intracellular vesicular cargo, reorients to a position just beneath the center of the IS. It has been proposed that these events focus secretion toward the target cell by bringing granules containing cytolytic factors close to the synaptic membrane. This model has not been rigorously tested, however. In addition, other possible roles for cytoskeletal dynamics at the IS, such as the transfer of mechanical signals to the target cell, remain unexplored. Over the past five years, we have defined two key mechanisms that shape the synaptic cytoskeleton, a diacylglycerol dependent pathway that guides the centrosome and a phosphoinositide 3-kinase (PI3K) dependent pathway that controls F-actin ring formation. We will now examine how these mechanisms contribute to CTL function. Our overall hypothesis is that centrosome polarization, central F-actin clearance, and peripheral F-actin dynamics together provide strength and specificity to CTL effector responses. The following Specific Aims will be pursued: 1) Determine the role of centrosome polarization in guiding CTL secretory responses; 2) Determine the mechanism and function of F-actin clearance; and 3) Determine how PI3K dependent force exertion potentiates cytotoxicity. For the first Aim, a novel genetic strategy based on conditional deletion of the scaffolding protein SAS4 will be used to remove the centrosome from CTLs. For the second Aim, gain- and loss-of-function experiments will be used to evaluate how actin- remodeling factors of the gelsolin family influence cytoskeletal polarization and CTL effector responses. For the third Aim, biophysical approaches will be used to quantify mechanotransduction at the IS and examine how force exertion potentiates target cell killing. This work is technically innovative because it incorporates state-of- the-art imaging modalities, genetic tools, and biophysical methods to explore lymphocyte function in new ways. It also advances innovative concepts about how the structure of the IS facilitates the transfer chemical and also mechanical information to the target cell. The proposed studies are important because they will lead to a comprehensive understanding of how synaptic architecture specifies CTL function, which will aid efforts to harness and control CTL activity in immunotherapeutic contexts. Hence, our proposal is relevant to the NIH mission in that it will contribute to the advancement of knowledge that could improve human health.